**6. Microarray for aflatoxin B1 detection**

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple analytes in a single experiment and consist of a biosensor *micro* o *nano* arrays.

Research on microarrays as multianalyte biosystems has generated increased interest in the last decade. The main feature of the microarray technology is the ability to simultaneously detect multiple analytes in one sample by an affinity-binding event at a surface interface. In some cases immunoanalytical microarrays have the potential to replace conventional chromatographic techniques. They are applied if the number of samples is high or analysis by current methods is difficult and/or expensive. Therefore, microarray platforms have a great potential as monitoring systems for the rapid assessment of water or food samples. Antibody-based microarrays are a powerful tool for analytical purposes, also for Aflatoxins detection application. Immunoanalytical microarrays are a quantitative analytical technique using antibodies as highly specific biological recognition elements. They can be designed for a variety of analytical applications producing rapid results with low limits of detection (LOD).

For these reasons in association to some biosensors for Aflatoxins examples, we reported in this Chapter also a feasibility study, made in our laboratories, on application of antibodies microarrays for simultaneous analysis of two different mycotoxins (Aflatoxin-B1 and Fumonisin B1). In this work we developed a competitive immunoassay in a microarray format and with the described method observed different microarray patterns in samples containing Aflatoxin-B1 or Fumonisine or either analytes at a ppb concentration range (Lamberti et al., 2009). The quality of the microarray data is comparable to data generated by a microplate-based immunoassay (ELISA), but further investigations are needed in order to better characterize these methods when applied for food contaminants determination. In any case we hope that our results can confirm the feasibility to develop hapten microarrays as for the immunochemical analysis of mycotoxins, as above described for others small organic molecules (e.g. bacterial toxins or biological warfare agents).

Enzyme linked immunosorbent assay (ELISA) and fluorescence immunoassay (FIA) are excellent survey tools for many analitycal purpose because of their high-throughput, user friendliness, and field portability. These important characteristics make immunoassays attractive tools for food testing by regulatory agencies to ensure food safety. Immunoassay is traditionally performed as individual test, however in many cases it is necessary to perform a panel of tests on each sample (detection of drug residues). To address this requirement, microarray-based immunoassay technologies have been developing utilizing microarray platform (multianalyte analysis) and classic immunoassay (multi-samples analysis).

In recent years, the antibody microarray technology has made significant progress, going from proof -of-concept designs to established high-performing technology platforms capable of targeting non-fractionated complex samples, as proteoma (Blohm & Guiseppi-Elie., 2001).

Microarrays consist of immobilized biomolecules spatially addressed on planar surfaces, microchannels or microwells, or an array of beads immobilized with different biomolecules.

Biosensors for Aflatoxins Detection 157

Fig. 7. Scheme of sensing assay for Aflatoxin B1 (Lamberti et al., 2009)

Mycotoxin analysis in food and feed is generally a multi-step process comprised of sampling, sample preparation, toxin extraction from the matrix (usually with mixtures of water and polar organic solvents), extract clean-up and finally detection and quantitative

**7. Conclusion** 

determination, for these reasons.

Biomolecules commonly immobilized on microarrays include oligonucleotides, polymerase chain reaction (PCR) products, proteins, lipids, peptides and carbohydrates. Ideally, the immobilized biomolecules must retain activity, remain stable, and not desorb during reaction and washing steps. The immobilization procedure must ensure that the biomolecules are immobilized at optimal density to the microarray surface for efficient binding (Venkatasubbarao, S. et al., 2004).

Some microarray applications are focused on current trends in the movement of this technology from being a purely research method to becoming an analytical instrument applicable in the clinic and as well as in human health (Koppal, T. , 2004).

According to this trend we have tried to transfer the immunoassay method from microtiter plates into a microarray format in order to develop a multiparametric, rapid, sensitive and inexpensive method for the the detection of mycotoxins for food safety application.

To perform our test and check the feasibility of this format, we focused our studies on the most popular mycotoxins Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1) and developed a competitive immunoassay in a microarray format, using the Dr.Chip platform provided by Life Line Lab Co. (Pomezia, Italy) and used also for other applications (Lamberti, 2010).

Microarray platform is equipment to create microarrays and to read the final results, via densitometric detection, based on the enzymatic and colorimetric assay. In Fig.6 are reported a detail of the plastic probe tray for protein spotting and pins. In the same picture is also shown the scheme of the glass treated with functional protein linker.

Fig. 6. Microarray spotting platform used for Aflatoxin B1 detection

As in other conventional competitive immunoassay the color intensity and corrispondent grey values obtained from antigen microarrays BSA-Afla B1, prepared as described in this paper and used in our immunological tests, are in inverse proportion to antigen concentration in standard solutions. Assay method for Aflatoxin is described in Fig.7.

Biomolecules commonly immobilized on microarrays include oligonucleotides, polymerase chain reaction (PCR) products, proteins, lipids, peptides and carbohydrates. Ideally, the immobilized biomolecules must retain activity, remain stable, and not desorb during reaction and washing steps. The immobilization procedure must ensure that the biomolecules are immobilized at optimal density to the microarray surface for efficient

Some microarray applications are focused on current trends in the movement of this technology from being a purely research method to becoming an analytical instrument

According to this trend we have tried to transfer the immunoassay method from microtiter plates into a microarray format in order to develop a multiparametric, rapid, sensitive and

To perform our test and check the feasibility of this format, we focused our studies on the most popular mycotoxins Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1) and developed a competitive immunoassay in a microarray format, using the Dr.Chip platform provided by Life Line Lab Co. (Pomezia, Italy) and used also for other applications (Lamberti,

Microarray platform is equipment to create microarrays and to read the final results, via densitometric detection, based on the enzymatic and colorimetric assay. In Fig.6 are reported a detail of the plastic probe tray for protein spotting and pins. In the same picture

inexpensive method for the the detection of mycotoxins for food safety application.

applicable in the clinic and as well as in human health (Koppal, T. , 2004).

is also shown the scheme of the glass treated with functional protein linker.

Fig. 6. Microarray spotting platform used for Aflatoxin B1 detection

As in other conventional competitive immunoassay the color intensity and corrispondent grey values obtained from antigen microarrays BSA-Afla B1, prepared as described in this paper and used in our immunological tests, are in inverse proportion to antigen concentration in standard solutions. Assay method for Aflatoxin is described in Fig.7.

binding (Venkatasubbarao, S. et al., 2004).

2010).

Fig. 7. Scheme of sensing assay for Aflatoxin B1 (Lamberti et al., 2009)

### **7. Conclusion**

Mycotoxin analysis in food and feed is generally a multi-step process comprised of sampling, sample preparation, toxin extraction from the matrix (usually with mixtures of water and polar organic solvents), extract clean-up and finally detection and quantitative determination, for these reasons.

Biosensors for Aflatoxins Detection 159

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The availability of rapid and reliable methods for "in field" determination of fungine contamination, as Aflatoxins and other MTXs (mycotoxins) identification in foods is an increasing need for human health and food safety purposes. Gas chromatographic (GC) methods for Aflatoxins detection are generally used, due to their low detection limits and high selectivity. Laboratories generally have to analyze a large number of samples requiring adequate storage conditions and time-consuming sample pre-treatment and preconcentration procedures. The establishment of efficient method for Aflatoxins detection is therefore of high importance and new trends consist in development portable and easy to use biosensors.

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Biosensor assays are rapid, easy to perform, and inexpensive and could be advantageous in comparison with ELISA or GC/MS for analysis on food, but in our opinion, further improvements of analytical parameters such as precision, accuracy, and detection limits (especially for Aflatoxins biosensor applications) are required.

#### **8. Acknowledgment**

The authors are grateful to Prof.Tibor Hianik of Nuclear Physics and Biophysics, Comenius University, Bratislava, Slovakia and to Prof.Caterina Tanzarella, University of RomaTre Department of Biology, Rome, Italy for their advice and scientific support.

#### **9. References**


The availability of rapid and reliable methods for "in field" determination of fungine contamination, as Aflatoxins and other MTXs (mycotoxins) identification in foods is an increasing need for human health and food safety purposes. Gas chromatographic (GC) methods for Aflatoxins detection are generally used, due to their low detection limits and high selectivity. Laboratories generally have to analyze a large number of samples requiring adequate storage conditions and time-consuming sample pre-treatment and preconcentration procedures. The establishment of efficient method for Aflatoxins detection is therefore of high importance and new trends consist in development portable and easy to

Recently, biosensors have seen a great development in the field of small molecules analytical determination and these methods are upon constant improvement also for MTXs and for

At this developmental stage, biosensors for Aflatoxins and MTX (Mycotoxin) detection could be very useful as a qualitative/semiquantitative "field test" for identifying "positive" samples, reducing the number of samples to be reanalyzed in the laboratory, according to

Biosensor assays are rapid, easy to perform, and inexpensive and could be advantageous in comparison with ELISA or GC/MS for analysis on food, but in our opinion, further improvements of analytical parameters such as precision, accuracy, and detection limits

The authors are grateful to Prof.Tibor Hianik of Nuclear Physics and Biophysics, Comenius University, Bratislava, Slovakia and to Prof.Caterina Tanzarella, University of RomaTre

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use biosensors.

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**8. Acknowledgment** 

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**9. References** 


**10** 

**Enzymatic Sensor for** 

Da-Ling Liu1,3 et al.\*

*P.R.China* 

**Sterigmatocystin Detection and** 

 *5IACM Laboratory, Macau,* 

**Feasibility Investigation of Predicting** 

*1Institute of Microbial Biotechnology, Jinan University, Guangzhou,* 

**Aflatoxin B1 Contamination by Indicator** 

 *2National Engineering Research Center of Genetic Medicine,Guangzhou, 3Guangdong Provincial Key Laboratory of Bioengineering Medicine,Guangzhou, 4Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou,* 

The development of fast and sensitive sensor for mycotoxins' detection has drawn a great attention in resent years (Prieto-Simom, B. et al., 2007). However, to construct anti-

Sterigmatocystin, a biogenic precursor of aflatoxin B1, has been classified as group 2B by the International Agency for Research on Cancer (IARC). Its chemical structure consists of a xanthone nucleus attached to bisfuran and it bears a close structural similar to aflatoxin B1 (Fig. 1) (Versilovskis et al., 2008). The toxicity of sterigmatocystin is primarily confined to the liver and kidney and closely correlated to the occurrence of hepatocellular carcinoma, gastric carcinoma and esophagus carcinoma (Purchase & van der Watt, 1970). Contamination of cereals with *Aspergillus* fungi refers to harmfulness, due to the potential of sterigmatocystin production by these fungi. Sterigmatocystin is similar to aflatoxin B1 both in the carcinogenicity and fluorescence excitability. While the fluorescence of sterigmatocystin is not so strong as aflatoxin B1 and the sterigmatocystin-antibody not commercially available, the detection of sterigmatocystin is harder or/and cost more. Several methods for the detection of sterigmatocystin have been established, including thinlayer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid

Hui-Yong Tan1, Jun-Hua Chen1,4, Ada Hang-Heng Wong1, Meng-Ieng Fong5, Chun-Fang Xie1,2,

**1. Introduction** 

 \*

**1.1 Enzymatic sensory detection of sterigmatocystin** 

Shi-Chuan Li1, Hong Cao1 and Dong-Sheng Yao1,2

*5 IACM Laboratory, Macau, P.R.China.* 

interference biosensor for the practical samples is still challenge.

*1 Institute of Microbial Biotechnology, Jinan University, Guangzhou, P.R.China, 2 National Engineering Research Center of Genetic Medicine,Guangzhou, P.R.China, 3 Guangdong Provincial Key Laboratory of Bioengineering Medicine,Guangzhou, P.R.China, 4 Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P.R.China,* 

